2.1 | Design and synthesis of functionalized
PEG-lipids
We designed and prepared PCNDs conjugated with cargo proteins and 9E5
using end-functionalized poly(ethylene glycol)(PEG)-lipids (Figure 1A
and 1B). The PCNDs used in this study consisted of an outer lipid
coating and an inner perfluorocarbon core. As previously
reported[30,31], dipalmitoyl phosphatidylcholine
(DPPC) and its PEGylated analog (DPPE-PEG-OMe) were used as the basic
lipid components (Figure 1B). Perfluorohexane (PFH) was employed as the
perfluorocarbon based on a pioneering report on PCND-mediated endosomal
rupture[28]. The lifetime of microbubbles
generated from PCNDs with the PFH core was on the order of microseconds,
and after ultrasound-induced vaporization, the bubbles were reported to
disappear within 10 μs by re-condensation[32].
Accordingly, in the reported endosomal rupture system, PCNDs were
assumed to vaporize only for a moment inside the cells, thereby avoiding
significant cell damage (Figure 1C)[28]. In our
previous studies[30,31], biotinylated antibody 9E5
was conjugated with biotinylated PEG-lipid (DPPE-PEG-biotin) by
crosslinking with a streptavidin analog, NeutrAvidin, on the lipid
coating of PCNDs (Figure 1B). Additionally, in this study, cargo
proteins were designed to conjugate onto the PCND surface via a
disulfide linker because they were required to be released in the
reductive intracellular environment. Therefore, a PEG-lipid
functionalized with 3-[(2-pyridyl) dithio] propionyl
(DPPE-PEG-PyDTP) moiety was synthesized (see Supporting Information,
Figure S1 and S2) and incorporated into the lipid coating of the PCNDs
(Figure 1A). In the present design, the thiol group of the proteins
reacted with DPPE-PEG-PyDTP, resulting in conjugation via
thiol-disulfide exchange (Figure 1A).